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Comparison of yeast cell protein solubilization procedures for two‐dimensional electrophoresis
Author(s) -
Harder Alois,
Wildgruber Robert,
Nawrocki Arek,
Fey Stephen J.,
Mose Larsen Peter,
Görg Angelika
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:4/5<826::aid-elps826>3.0.co;2-a
Subject(s) - lysis , chemistry , lysis buffer , tris , chromatography , urea , sonication , thiourea , sodium dodecyl sulfate , chaps , dilution , bradford protein assay , yeast , gel electrophoresis , biochemistry , solubilization , organic chemistry , physics , thermodynamics
Abstract Three different procedures for the solubilization of yeast ( S. cerevisiae ) cell proteins were compared on the basis of the obtained two‐dimensional (2‐D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with “standard” lysis buffer (9 M urea, 2% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS)), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 m M tris(hydroxymethyl)aminomethane (Tris)‐HCl, pH 7.0, followed by dilution with “standard” lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea / 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high M r proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high M r proteins were further improved by pre‐boiling with SDS and using thiourea/urea lysis buffer instead of “standard” lysis buffer (procedure iii).